Figure. Pedigrees of the 4 families segregating the C9orf72 hexanucleotide repeat expansion. Black diamonds indicate individuals with amyotrophic lateral sclerosis (ALS); green diamonds, individuals with frontotemporal dementia (FTD); red diamonds, individuals with both ALS and FTD; gray diamonds, individuals with preliminary signs of dementia; and white diamonds, unaffected or at-risk individuals. W indicates wild-type alleles; M, mutant alleles. Individuals with diagonal lines are deceased. Not all family members are shown to protect privacy.
Daoud H, Suhail H, Sabbagh M, et al. C9orf72 hexanucleotide repeat expansions as the causative mutation for chromosome 9p21–linked amyotrophic lateral sclerosis and frontotemporal dementia. Arch Neurol.Published online May 21, 2012. doi:10.1001/archneurol.2012.377.
eFigure. Repeat-primed PCR fragment analysis with the GeneMapper software. Electropherograms are zoomed to 800 relative fluorescence units. Numbers above the traces indicate the fragment’s lengths in base pairs. The grey vertical bars indicate the allele sizes, with the first bar indicating three GGGGCC hexanucleotide repeats. One repeat carrier from each family and one unaffected individual are represented. A typical saw-tooth pattern with a 6 bp periodicity is observed in affected individuals.
Daoud H, Suhail H, Sabbagh M, Belzil V, Szuto A, Dionne-Laporte A, Khoris J, Camu W, Salachas F, Meininger V, Mathieu J, Strong M, Dion PA, Rouleau GA. C9orf72 Hexanucleotide Repeat Expansions as the Causative Mutation for Chromosome 9p21–Linked Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Arch Neurol. 2012;69(9):1159-1163. doi:10.1001/archneurol.2012.377
Author Affiliations: CHUM Research Center and Department of Medicine, Centre of Excellence in Neuroscience of Université de Montréal (Drs Daoud, Suhail, Sabbagh, Dion, and Rouleau and Mss Belzil, Szuto, and Dionne-Laporte), and Department of Pathology and Cell Biology, Faculty of Medicine (Dr Dion), Université de Montréal, and Research Center, CHU Sainte-Justine (Dr Rouleau), Montreal, and Clinique des maladies neuromusculaires, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke (Dr Mathieu), Quebec, and Department of Clinical Neurological Sciences and Robarts Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario (Dr Strong), Canada; and Unité de Neurologie Comportementale et Dégénérative, Institute of Biology, Montpellier (Dr Khoris and Camu), and Fédération des Maladies du Système Nerveux, Assistance Publique–Hôpitaux de Paris, Centre de référence maladies rares SLA, Hôpital Pitié-Salpêtrière, Paris (Drs Salachas and Meininger), France.
Objective To further assess the presence of a large hexanucleotide repeat expansion in the first intron of the C9orf72 gene identified as the genetic cause of chromosome 9p21–linked amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) in 4 unrelated families with a conclusive linkage to c9ALS/FTD.
Design A repeat-primed polymerase chain reaction assay.
Setting Academic research.
Participants Affected and unaffected individuals from 4 ALS/FTD families.
Main Outcome Measure The amplified C9orf72 repeat expansion.
Results We show that the repeat is expanded in and segregated perfectly with the disease in these 4 pedigrees.
Conclusion Our findings further confirm the C9orf72 hexanucleotide repeat expansion as the causative mutation for c9ALS/FTD and strengthen the hypothesis that ALS and FTD belong to the same disease spectrum.
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are adult-onset neurodegenerative diseases generally characterized by a rapid progression after symptom onset. Amyotrophic lateral sclerosis is the most prevalent motor neuron disease worldwide, where the selective degeneration of upper and lower motor neurons in the brain and spinal cord lead to progressive paralysis and death, typically of respiratory failure within 3 to 5 years of symptom onset.1 Frontotemporal dementia is the second most common form of presenile dementia after Alzheimer disease and is characterized by the degeneration of neurons in the frontal and anterior temporal lobes leading to behavioral dysfunction and impairment in executive functions and language.2 Interestingly, up to 15% of patients with ALS have FTD,3 and a further 30% have evidence of cognitive impairment, suggesting that these 2 conditions have a common genetic background.
Although most ALS cases are sporadic, approximately 5% are familial and show a mendelian pattern of inheritance.4 Of these, the most frequently identified cause is mutations in the SOD1 gene5 (15%-20%), followed by the TARDBP6 and FUS7 genes (1%-3% each). Additionally, mutations in other genes have been linked to familial ALS but these appear to be very rare.8 On the other hand, up to 50% of patients with FTD have family members with dementia and/or cognitive and behavioral changes.9 Similarly, genetic variations in the MAPT10 and PRGN11 genes explain almost 50% of familial FTD cases and are considered as the main genetic cause of FTD. Variations in other genes, including CHMP2B, VCP, TARDBP, and FUS, contribute to less than 5% of all FTD cases.12
Growing evidence indicates that ALS and FTD are 2 phenotypic manifestations of a common underlying genetic cause. The identification of ubiquitinated TDP-43–positive inclusions as a common pathological hallmark of patients with ALS and FTD first contributed to the merging of the ALS and FTD fields.13 Most importantly, the co-occurrence of ALS and FTD within the same families and sometimes within the same individuals was repeatedly reported, particularly over the last 5 years, strongly implicating common genetic components for these 2 conditions. Indeed, linkage analyses of extended pedigrees in which both ALS and FTD segregate have led to the identification of a very robust locus on the chromosome 9p21.14- 16 Very recently, a large hexanucleotide repeat expansion in the first intron of the C9orf72 gene has been identified as the genetic cause of chromosome 9p21-linked ALS/FTD (c9ALS/FTD) in families and the most common cause of familial ALS and FTD to date.17- 19
We have previously reported 3 families with ALS/FTD from Canada and France with evidence of linkage to chromosome 9p21.16 Herein, we show that the hexanucleotide repeat expansion in C9orf72 is the underlying genetic defect in these 3 families as well as a fourth family from Quebec, Canada, and that this expansion segregated perfectly with the disease in these 4 families.
Four unrelated families including patients with ALS/FTD were included in this study (Figure). Three of them (Que-1, Que-23, and Fr-104) were previously linked to the c9ALS/FTD locus.16 Informed written consent was obtained from all participating family members, and the study was approved by the ethics committee of the Centre Hospitalier de l’Université de Montréal, Montreal, Quebec. Patients with ALS met the diagnosis of definite or probable ALS as defined in the El Escorial criteria.20 All patients with FTD exhibited cerebral atrophy at neuroimaging. Some individuals had preliminary signs of dementia; others had only FTD without any motor involvement (Figure). The clinical descriptions of the Que-1, Que-23, and Fr-104 families have been previously reported.16 Briefly, these 3 pedigrees contain 32 affected individuals, 16 with ALS, 4 with FTD, 7 with ALS/FTD, and 5 with dementia. Since the first description of these 3 families,16 11 additional individuals became affected with ALS, FTD, or both (8 new cases in Que-1 and 3 in Que-23). The fourth pedigree (Que-364) contains 2 individuals with ALS, 1 with FTD, and 1 with dementia.
Genomic DNA was extracted from blood samples or lymphoblastoid cell lines using standard methods. To assess the presence of an expanded hexanucleotide repeat in the C9orf72 gene, we performed a repeat-primed polymerase chain reaction (PCR) assay using the FastStart PCR Master Mix (Roche) and the previously optimized assay conditions.17,18 The repeat was amplified in all affected and unaffected individuals from these 4 pedigrees for whom DNA was available. The PCR products were analyzed on an ABI 3730 sequencer with GeneMapper software version 4.0 (Applied Biosystems). Individuals carrying the expansion showed a characteristic pattern with a 6–base pair periodicity (eFigure).
We amplified the C9orf72 hexanucleotide repeat in 4 unrelated families including patients with ALS and/or FTD (Figure). Using the repeat-primed PCR method, we show that this repeat is expanded and segregated with the disease in these 4 pedigrees. We also show that some at-risk individuals carry the C9orf72 expansion but did not develop any disease signs, probably because they are still younger than the age at onset (Figure). This expansion was absent from 190 French Canadian neurologically healthy individuals (data not shown). Altogether, 36 affected individuals carry the expansion: 18 with ALS (50.0%), 5 with FTD (13.8%), 7 with ALS/FTD (19.4%), and 6 with preliminary signs of dementia (16.6%). The average age at onset in 22 patients for whom clinical records were available was 59.9 years (range, 46-81 years) with an average disease duration of 3.3 years (range, 2-5 years) (Table). The site of onset of ALS was bulbar in 6 patients and spinal in 9 patients. In family Que-1, the average age at onset was 48 years in the fifth generation, which was younger than that of 8 patients from the fourth generation (65 years; 95% CI, 60.2-69.9) and that of the third generation (81 years).
In 2007, our group reported 3 relatively large ALS/FTD pedigrees with genetic linkage to chromosome 9p21.16 In this study, we report the hexanucleotide repeat expansion in the noncoding region of the C9orf72 gene as the underlying genetic cause of ALS/FTD in these 3 families as well as a newly recruited family with ALS/FTD. We show that the repeat expansion segregates perfectly with the disease in these 4 families and is absent from a cohort of French Canadian controls (data not shown), suggesting that this expansion is fully penetrant.
However, given that the repeat-primed method we and other groups have used17- 19 cannot establish the exact sizes of the expanded alleles, we could not establish a genotype-phenotype correlation and determine the link between allele sizes and disease severity. For now, we only noticed a trend toward younger age at onset in 3 generations of the family Que-1. Although clinical information was not available in all affected individuals of this family, this trend suggests the presence of anticipation in this family that could possibly be explained by the instability of this hexanucleotide, as its number may increase over the generations. However, we did not observe this anticipation in the other 3 families, which could be related to the small size of these families, notably Fr-104 and Que-364, or the lack of clinical information.
The other caveat of the repeat-primed assay is that the exact threshold leading to disease could not be established. Indeed, the development of other methods that can accurately size the large alleles, such as Southern blotting or long-range PCR assays, is needed to better understand the correlation between the allele sizes and disease parameters such as age at onset, site of onset, and disease duration. This accurate sizing is also essential to assess whether this repeat is unstable and whether this instability occurs on the paternal or the maternal alleles.
In summary, we report herein the hexanucleotide expansion in the C9orf72 gene as the genetic cause of c9ALS/FTD in 4 families with a French and French Canadian ethnicity. Our data further confirm this hexanucleotide repeat expansion as the causative mutation for c9ALS/FTD. Although the mechanism by which these large expansions lead to neurodegeneration has yet to be identified, the identification of the C9orf72 gene as a common cause of ALS and FTD strengthens the hypothesis that these diseases are 2 phenotypic ends to the same spectrum and suggests that additional genes causing both ALS and FTD are likely to be identified in the future. Altogether, these newly identified genes would increase our understanding of these diseases and hopefully lead to the development of therapeutic strategies.
Correspondence: Guy A. Rouleau, MD, PhD, FRCPC, Centre of Excellence in Neuroscience, CHUM Research Center and Department of Medicine, Université de Montréal, 2099, Alexandre De-Seve Street, Room Y-3633, Montreal, QC H2L 2W5, Canada (firstname.lastname@example.org).
Accepted for Publication: February 21, 2012.
Published Online: May 21, 2012. doi:10.1001/archneurol.2012.377
Authors Contributions:Study concept and design: Daoud, Dion, and Rouleau. Acquisition of data: Daoud, Suhail, Sabbagh, Belzil, Szuto, Dionne-Laporte, Khoris, Camu, Salachas, Meininger, Mathieu, and Strong. Analysis and interpretation of data: Daoud and Salachas. Drafting of the manuscript: Daoud, Sabbagh, Belzil, and Salachas. Critical revision of the manuscript for important intellectual content: Suhail, Szuto, Dionne-Laporte, Khoris, Camu, Meininger, Mathieu, Strong, Dion, and Rouleau. Statistical analysis: Salachas. Obtained funding: Dion and Rouleau. Administrative, technical, and material support: Suhail, Szuto, Dionne-Laporte, Khoris, Camu, Salachas, Meininger, Mathieu, and Rouleau. Study supervision: Rouleau.
Financial Disclosure: None reported.
Funding/Support: This work was financially supported by the ALS Division of the Muscular Dystrophy Association, the Frick Foundation for ALS Research, and the Canadian Institutes of Health Research. Dr Daoud is supported by a postdoctoral fellowship from the ALS Society of Canada and the Canadian Institutes of Health Research. Dr Daoud was also supported by a postdoctoral fellowship from the ALS Association and the Fonds de Recherche en Santé du Québec. Dr Rouleau holds a Canada Research Chair in Genetics of the Nervous System and the Jeanne et J-Louis-Lévesque in Genetics of Brain Diseases.
Additional Contributions: We thank the subjects and their parents for their participating in this study. Annie Levert, DEC, provided technical assistance.